Proteolytic modification of the extracellular environment is essential for physiological tissue remodeling, as well as the progression of a number of diseases, including cancer. The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated serine proteases, in particular, their relation to the development, regeneration, and malignant transformation of keratinized epithelium. Plg activation in tumor progression The plasminogen activation (PA) system is a sophisticated system of serine proteases, protease inhibitors, and protease receptors that governs the conversion of the abundant protease zymogen, plasminogen (Plg), to the active, multifunctional protease, plasmin. We found that Plg promotes the orthotopic progression of fibrosarcoma in mice, and that host Plg and tumor cell-produced Plg activator cooperate to promote fibrosarcoma progression independently of host Plg activator, Plg activator inhibitor, and urokinase Plg activator receptor (uPAR). Furthermore, we have linked tumor cell Plg activation to the suppression of the accumulation of tumor-infiltrating macrophages, and to the promotion of tumor angiogenesis. Initiation of the urokinase (uPA)-mediated Plg activation cascade Attempts to identify the initiation routes for the proteolytic activation of the inactive pro-form of uPA (pro-uPA) in vivo have been hampered by the lack of reagents capable of detecting the state of activation of the minute quantities of uPA that are present in vivo. In collaboration with the BTTS, OIIB, we have developed modified anthrax toxins that can be used to specifically detect cell surface uPA activity in vivo, and have used these reagents to perform the first analysis of the biochemical requirements for productive pro-uPA activation in vivo. The studies have established unequivocally that uPAR is critical for generating cell surface uPA activity in vivo, that plasmin is critical in vivo for the efficient conversion of pro-uPA to active uPA, both in the context of physiological Plg activation and on the tumor cell surface, and that Plg activator inhibitor-1 restricts the continuous generation of active cell surface uPA in vivo. Plasmin substrates in epidermal remodeling Plg-deficiency impairs both squamous cell carcinoma progression and skin wound healing. In collaboration with the MMPU, we have found that the dissolution of fibrillar collagen by keratinocytes requires both cell surface Plg activation and matrix metalloprotease activity. Furthermore, we have shown that cell surface Plg and either tissue Plg activator or urokinase Plg activator are absolutely required for the activation of the fibrillar collagenase matrix metalloproteinase-13 by keratinocytes. Our findings suggest that Plg activation facilitates keratinocyte-mediated collagen breakdown via the direct activation of matrix metalloproteinase-13 and possibly other fibrillar collagenases. The novel pathway underscores the likely existence of multiple, cell type and context-specific pathways for collagen dissolution, and may be relevant to physiological remodeling of the skin, oral cavity, and urogenital tract, as well as to squamous cell carcinoma progression. The uPAR-Associated Protein, uPARAP The uPAR-associated protein, uPARAP, is a novel transmembrane glycoprotein that associates with ligand-occupied uPAR. We have shown that uPARAP is expressed at sites of active tissue remodeling during development, is widely expressed in adult mesenchymal tissues, and is expressed in many malignant tissues including squamous cell carcinoma. We have generated mice with a targeted deletion of the uPARAP gene and, surprisingly, found that uPARAP is required for the cellular uptake of collagen. Fibroblasts deficient in uPARAP displayed a near complete abrogation of the endocytosis of a wide range of collagen types. Furthermore, uPARAP-deficient cells showed a diminished adhesion to many different collagen species, and impaired migration on fibrillar collagen. Collectively, these studies have identified uPARAP as a key component in cellular interactions with collagen. We are currently determining the function of uPARAP in matrix turnover during carcinoma progression. Urokinase-activated anthrax toxins The acquisition of cell surface urokinase plasminogen activator activity is a hallmark of human malignancy. In collaboration with the BTTS, OIIB, we generated an engineered anthrax toxin that is specifically activated by cell surface uPA by replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase. This mutation conferred cell surface uPA-dependent toxin activation in vivo, as determined using a panel of plasminogen, plasminogen activator, - receptor, and - inhibitor-deficient mice. The engineered toxin displayed limited toxicity to normal tissue but potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin, and could eradicate established solid tumors. This tumoricidal activity was strictly dependent on tumor cell surface PA. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent. Unexpected roles of the PA system in brain function Tissue-type plasminogen activator (tPA) is expressed in the CNS during events that require neuronal plasticity. In collaboration with Dr. Daniel Lawrence, American Red Cross, we found that tPA is essential for seizure progression by promoting the synchronization of neuronal activity required for seizure spreading. Surprisingly, we found that this activity of tPA was Plg-independent, as Plg-deficient mice displayed normal seizure spreading. These findings indicate that tPA acts on a substrate different from Plg to promote seizure progression and likely other physiological and pathological processes in the CNS. Novel cell surface serine proteases in epidermal development, repair and malignancy In collaboration with the MCU, OPCB, we applied laser capture microdissection, cDNA array analysis, and in situ hybridization to identify novel serine proteases associated with skin wound healing and squamous cell carcinogenesis. This analysis led to the identification of the type II transmembrane serine protease, matriptase, as a protease that is expressed by reepithelializing wound keratinocytes, in squamous cell carcinoma and in other malignancies. By introducing a null mutation into the mouse matriptase gene, we have found that this novel membrane serine protease has pleiotropic functions in epidermal development. Loss of matriptase was associated with profound malformations of the stratum corneum, seriously compromised epidermal barrier function, and caused uniform perinatal lethality due to uncontrolled dehydration. Furthermore, hair follicle development was severely affected with generalized follicular hypoplasia and dysmorphism, hair canal agenesis, and follicular abortion. We are currently unraveling the specific functions of matriptase in epidermal development, and are determining the contribution of matriptase to skin wound healing and squamous cell carcinogenesis. Furthermore, we have initiated a collaboration with Dr. Toni Antalis, The American Red Cross, to take advantage of the recently available mouse and human genome databases to identify additional membrane serine proteases potentially involved in epidermal malignancy. Currently, we are investigating the expression and function of five novel matriptase-related proteases in epidermal biology and pathology.